All living bodies are comprised of individual cells, each cell defining an environment where various biological and chemical reactions take place. In particular, each cell contains a cell membrane that separates the internal environment of the cell from the external environment and thereby controls the entry and exit of various nutrients and waste. Additionally, the cell membrane includes various proteins, sugars, and other molecules that “identify” a particular cell type, these identifying molecules commonly being referred to as antigens.
In order to better understand the function and pathologies of cells, numerous methods have been developed to isolate and concentrate a desired target cell population from a mixed cell population so that the target cell population can be further analyzed. One such method is based upon cell density wherein a mixture of cells is spun at high speeds in a centrifuge so that the higher density cells become separated for the lesser density cells. Although this method is effective at separating different cells, centrifugation does not have good cell-separation specificity as different types of cells may have the same or similar cell density.
Accordingly, more sophisticated cell separation techniques have been developed wherein cells are separated based upon the presence of certain cellular identifiers, namely, antigens, found on the cellular membrane. More specifically, these selection methods are based upon using antibodies that react with antigens found on a particular target cell membrane. In one such method, the antibodies are affixed on the surface of a substrate, such as magnetic beads or small iron-coated particles. When mixed with the cell sample, the antibody-coated beads or particles bind to the specific antigens on the cell membrane. As a sample cell solution is passed through a magnetic separation column, the magnetic particles with the target cells attached then bind to the surface of the magnetic field. The target cells are then released from the column by removing the magnetic field from the cell separation column. Other known methods use variations of target cell binding in continuous-flow “immunoaffinity” columns. Generally, with immunoaffinity columns, once the target cells bind to the column by antigen-antibody affinity, the bound target cells are released by mechanically agitating the immunoaffinity column.
Cell separation techniques based upon cellular membrane identifiers are particularly useful in isolating specific cells as such techniques may be modified or tailored for specific target cells. Indeed, such highly specific cell separation techniques are particularly useful for diagnosing and treating specific and potent diseases such as, but not limited to, autoimmune diseases or cancer.
The utility of immunoseparation techniques as a diagnostic tool is evident given the prevalence of various diseases. Cancer, for instance, is expected to afflict approximately 1.3 million people in 2002 and result in approximately 500,000 deaths. Studies have shown, however, that early detection of cancer results in improved survival rates as treatment is more likely to be successful during the early stages of cancer. Yet while early diagnosis and treatment increases the chances of survival, there still remains the possibility of relapse. Accordingly, there has been considerable research into the causes of cancer relapse.
In particular, over the past 12 years, numerous research studies have been designed to track the presence of low numbers of micrometastic tumor cells (so called “micrometastases”) in blood, bone marrow, and effusion fluids in patients with cancer. Studies have shown that the presence of tumor micrometastases in blood and bone marrow at time of surgery is a strong prognostic indicator of poor prognosis and early relapse in breast, prostate, ovarian, and lung cancer patients. Furthermore, the reappearance of circulating tumor cells following chemotherapy appears to herald the earliest indication of disease recurrence. Accordingly, the early detection of these micrometastases may result in higher survival rates for patients in relapse.
While the presence of micrometastases are strong indicators of cancer, these tumor cells are particularly difficult to detect as the reported frequency of micrometastatic tumor cells range from 1-5 micrometastatic tumor cells per 100,000-1,000,000 bone marrow cells and from 1 micrometastatic tumor cell in 1,000,000 to 100,000,000 blood cells. Despite the low frequency of micrometastases, various methods have been developed to concentrate or isolate the micrometastatic cells from blood, bone marrow, or effusion fluids using immunoselection methods such as, but not limited to, immunomagnetic separation/isolation, immunocolloidal separation/isolation, or flow cytometric separation/isolation.
While these prior art immunoselection methods have proven useful, these methods can be inefficient as they require considerable operator intervention during the separation process. For instance, the separation column usually needs cleaning and priming prior to the introduction of a sample solution. Furthermore, the column requires constant monitoring during the separation process. As a result, the efficiency, accuracy, and recovery of targeted cell is often directly related to operator skill or error. Accordingly, it is desirable to have a cell separation device that minimizes operator error.
Moreover, the design of prior art immunoseparation columns may also hinder the recovery of a targeted cell. For instance, immunomagnetic separation/isolation methods result in permanent or semi-permanent adherence of magnetic beads/particles to the isolated cells. Accordingly, the difficulty and sometimes inability to remove the target cells from the magnetic beads reduces the accuracy of these methods. For example, isolated target cells may become damaged when the cells are separated from the column as relatively harsh chemical or mechanical processes are typically required to remove the target cells from the beads. This is particularly problematic when attempting to detect cells, such as micrometastases, which have a low frequency.
Furthermore, target cell recovery is predicated on having the proper target cell to magnetic bead ratio. If the target cell to bead ratio is not properly optimized, “background” interference may develop due to the presence of beads or particles that are not bound to the target cells thereby reducing the method's accuracy. However, optimizing the target cell to bead ratio is difficult as the frequency of the target cell is usually unknown.
Accordingly, there remains a need for devices and methods that optimize target cell isolation, purity, and viability. There also remains a need for devices and methods that isolate viable, uncompromised cells (physically and/or biochemically) so as to enable subsequent analysis and potential therapeutic applications of the isolated cells.